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Protein kinase A activates protein phosphatase 2A by phosphorylation of the B56delta subunit.

ABSTRACT: Our previous studies of DARPP-32 in striatal slices have shown that activation of D1 receptors leads to cAMP-dependent dephosphorylation of Thr-75, the Cdk5 site in DARPP-32. In the current study, we have elucidated a mechanism whereby protein phosphatase 2A (PP2A) is activated by a cAMP/PKA-dependent pathway, leading to dephosphorylation of Thr-75. PP2A consists of a catalytic C subunit that associates with the scaffolding A subunit and a variety of B subunits. We have found that the A/C subunits of PP2A, in association with the B56delta (or PPP2R5D) regulatory subunit, is an active DARPP-32 phosphatase. The B56delta subunit expressed in HEK293 cells forms a heterotrimeric assembly that catalyzes PKA-mediated dephosphorylation at Thr-75 in DARPP-32 (also cotransfected into HEK293 cells). The B56delta subunit is phosphorylated by PKA, and this increases the overall activity of PP2A in vitro and in vivo. Among four PKA-phosphorylation sites identified in B56delta in vitro, Ser-566 was found to be critical for the regulation of PP2A activity. Moreover, Ser-566 was phosphorylated by PKA in response to activation of D1 receptors in striatal slices. Based on these studies, we propose that the B56delta/A/C PP2A complex regulates the dephosphorylation of DARPP-32 at Thr-75, thereby helping coordinate the efficacy of dopaminergic neurotransmission in striatal neurons. Moreover, stimulation of protein phosphatase activity by this mechanism may represent an important signaling pathway regulated by cAMP in neurons and other types of cell.

SUBMITTER: Ahn JH

PROVIDER: S-EPMC1815292 | biostudies-literature | 2007 Feb

REPOSITORIES: biostudies-literature

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Protein kinase A activates protein phosphatase 2A by phosphorylation of the B56delta subunit.

Ahn Jung-Hyuck JH McAvoy Thomas T Rakhilin Sergey V SV Nishi Akinori A Greengard Paul P Nairn Angus C AC

Proceedings of the National Academy of Sciences of the United States of America 20070214 8

Our previous studies of DARPP-32 in striatal slices have shown that activation of D1 receptors leads to cAMP-dependent dephosphorylation of Thr-75, the Cdk5 site in DARPP-32. In the current study, we have elucidated a mechanism whereby protein phosphatase 2A (PP2A) is activated by a cAMP/PKA-dependent pathway, leading to dephosphorylation of Thr-75. PP2A consists of a catalytic C subunit that associates with the scaffolding A subunit and a variety of B subunits. We have found that the A/C subuni ...[more]

PMID: 17301223

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Project description:BackgroundType 2A protein phosphatase (PP2A) enzymes are serine/threonine phosphatases which comprise a scaffold A subunit, a regulatory B subunit and a catalytic C subunit, and have been implicated in the dephosphorylation of multiple cardiac phosphoproteins. B subunits determine subcellular targeting, substrate specificity and catalytic activity, and can themselves be regulated by post-translational modifications. We explored potential β-adrenergic regulation of PP2A in cardiomyocytes through phosphorylation of the regulatory B subunit isoform B56δ.Methods and resultsPhosphate affinity SDS-PAGE and immunoblot analysis revealed increased phosphorylation of B56δ in adult rat ventricular myocytes (ARVM) exposed to the β-adrenergic receptor (βAR) agonist isoprenaline (ISO). Phosphorylation of B56δ occurred at S573, primarily through stimulation of the β1AR subtype, and was dependent on PKA activity. The functional role of the phosphorylation was explored in ARVM transduced with adenoviruses expressing wild type (WT) or non-phosphorylatable (S573A) B56δ, fused to GFP at the N-terminus. C subunit expression was increased in ARVM expressing GFP-B56δ-WT or GFP-B56δ-S573A, both of which co-immunoprecipitated with endogenous C and A subunits. PP2A activity in cell lysates was increased in response to ISO in ARVM expressing GFP-B56δ-WT but not GFP-B56δ-S573A. Immunoblot analysis of the phosphoproteome in ARVM expressing GFP-B56δ-WT or GFP-B56δ-S573A with antibodies detecting (i) phospho-serine/threonine residues in distinct kinase substrate motifs or (ii) specific phosphorylated residues of functional importance in selected proteins revealed a comparable phosphorylation profile in the absence or presence of ISO stimulation.ConclusionsIn cardiomyocytes, βAR stimulation induces PKA-mediated phosphorylation of the PP2A regulatory subunit isoform B56δ at S573, which increases associated PP2A catalytic activity. This is likely to regulate the phosphorylation status of specific B56δ-PP2A substrates, which remain to be identified.

Dataset Information

Protein kinase A activates protein phosphatase 2A by phosphorylation of the B56delta subunit.

Publications

Protein kinase A activates protein phosphatase 2A by phosphorylation of the B56delta subunit.

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